Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens

Wang, Rui; Shu, Xian; Zhao, Huiwei; Xue, Qiong; Liu, Chao; Wu, Aici; Cheng, Feiyue; Wang, Lingyun; Zhang, Yihan; Feng, Jie; Wu, Nannan; Li, Ming

doi:10.1038/s41467-023-37789-y

Cited by 18 publications

(15 citation statements)

References 35 publications

(65 reference statements)

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“…1c), implying that its overexpression should be toxic, at least to E. coli cells. Thereby, we presumed that this toxic protein might be normally suppressed by CreA (and the Csy complex) in A. baylyi ADP1 cells, similar to the previously reported CRISPR-repressed RNA toxins within the CreTA modules 16,18 . To denote its putative proteinic nature, we hereinafter designated this KilAC domain-containing toxin as CreP for ‘CRISPR-repressed proteinic toxin’.…”

Section: Resultssupporting

confidence: 82%

“…Thus far, three CreTA modules, respectively from the I-B CRISPR-Cas of H. hispanica ATCC 33960 16 , the I-B CRISPR-Cas of H. hubeiense JI20-1 17 , and the I-F CRISPR-Cas of Acinetobacter sp. WCHA45 18 , have been experimentally investigated. Intriguingly, although their creT genes each has a mini ORF, it has been demonstrated that the toxic element is not their protein but rather their RNA products.…”

Section: Resultsmentioning

confidence: 99%

“…WCHA45 acts as a bactericidal RNA, with its detailed mechanism yet to be elucidated 18 . Due to the lack of reported Acr proteins for these CRISPR systems, we first sought to explore additional CreTA modules from I-F systems in this study to investigate their potential anti-anti-CRISPR role (type I-F Acr proteins have been extensively discovered and characterized).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

Shu,

Wang,

et al. 2024

Preprint

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Prokaryotic CRISPR-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here, we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally reppressed by the CRISPR-Cas effector, but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system, and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to non-target DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors, which illuminates that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders, no matter they are CRISPR-targeted or not.

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

Shu,

Wang,

et al. 2024

Preprint

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“…Given that DarTG is associated with mobile genetic elements and other phage defense systems 30,31 , perhaps it serves a dual purpose in phage defense and protection of the defense element that encodes it. In this way, it could potentially protect other defense systems somewhat similar to how the TA system CreTA safeguards associated CRISPR-Cas immune systems 52 .…”

Section: Discussionmentioning

confidence: 99%

Sporadic phage defense in epidemicVibrio choleraemediated by the toxin-antitoxin system DarTG is countered by a phage-encoded antitoxin mimic

Patel,

Seed

2023

Preprint

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Bacteria and their viral predators (phages) are constantly evolving to subvert one another. Many bacterial immune systems that inhibit phages are encoded on mobile genetic elements that can be horizontally transmitted to diverse bacteria. Despite the pervasive appearance of immune systems in bacteria, it is not often known if these immune systems function against phages that the host encounters in nature. Additionally, there are limited examples demonstrating how these phages counter-adapt to such immune systems. Here, we identify clinical isolates of the global pathogenVibrio choleraeharboring a novel genetic element encoding the bacterial immune system DarTG and reveal the immune system’s impact on the co-circulating lytic phage ICP1. We show that DarTG inhibits ICP1 genome replication, thus preventing ICP1 plaquing. We further characterize the conflict between DarTG-mediated defense and ICP1 by identifying an ICP1-encoded protein that counters DarTG and allows ICP1 progeny production. Finally, we identify this protein as a functional antitoxin that abrogates the toxin DarT likely through direct interactions. Following the detection of the DarTG system in clinicalV. choleraeisolates, we observed a rise in ICP1 isolates with the functional antitoxin. These data highlight the use of surveillance ofV. choleraeand its lytic phages to understand the co-evolutionary arms race between bacteria and their phages in nature.ImportanceThe global bacterial pathogenVibrio choleraecauses an estimated 1 to 4 million cases of cholera each year. Thus, studying the factors that influence its persistence as a pathogen is of great importance. One such influence is the lytic phage ICP1, as once infected by ICP1,V. choleraeis destroyed. To date, we have observed that the phage ICP1 shapesV. choleraeevolution through the flux of anti-phage bacterial immune systems. Here, we probe clinicalV. choleraeisolates for novel anti-phage immune systems that can inhibit ICP1 and discover the toxin-antitoxin system DarTG as a potent inhibitor. Our results underscore the importance ofV. choleraeand ICP1 surveillance to elaborate novel means by whichV. choleraecan persist in both the human host and aquatic reservoir in the face of ICP1.

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“…the loss or mutation of target AR genes. In the event of the pathogens deactivating CRISPR-Cas through various anti-CRISPR mechanisms, CreTA acts as a backup system by inducing cellular death, creating a dilemma for the target pathogen 9 (Figure 1). The authors named this innovative approach "ATTACK" (AssociaTe Toxin-Antitoxin and CRISPR-Cas to Kill Pathogens).…”

mentioning

confidence: 99%

A new technique to ATTACK the silent pandemic of antimicrobial resistance

Zhu

2023

mLife

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Associate toxin-antitoxin with CRISPR-Cas to kill multidrug-resistant pathogens

Cited by 18 publications

References 35 publications

CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements

Sporadic phage defense in epidemicVibrio choleraemediated by the toxin-antitoxin system DarTG is countered by a phage-encoded antitoxin mimic

A new technique to ATTACK the silent pandemic of antimicrobial resistance

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